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Inhibition of the 3′→ 5′ Exonuclease of Human DNA Polymerase ε by Fludarabine-terminated DNA

1996; Elsevier BV; Volume: 271; Issue: 32 Linguagem: Inglês

10.1074/jbc.271.32.19428

1083-351X

Ken-ichi Kamiya, Peng Huang, William Plunkett,

HIV/AIDS drug development and treatment

Incorporation of the anticancer drug fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate; F-ara-AMP) into the 3′-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3′-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3′→ 5′-exonuclease activity of DNA polymerase ε from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase ε was unable to effectively remove F-ara-AMP from the 3′-end of the oligomer. However, 3′-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase ε has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3′-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3′-5′ phosphodiester bond between F-ara-AMP at the 3′-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase ε, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase ε to the reaction. Furthermore, the addition of DNA polymerase α and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase ε had been incubated with 3′-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3′-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3′→ 5′ exonuclease of human DNA polymerase ε can remove 3′-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex." Incorporation of the anticancer drug fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate; F-ara-AMP) into the 3′-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3′-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3′→ 5′-exonuclease activity of DNA polymerase ε from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase ε was unable to effectively remove F-ara-AMP from the 3′-end of the oligomer. However, 3′-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase ε has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3′-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3′-5′ phosphodiester bond between F-ara-AMP at the 3′-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase ε, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase ε to the reaction. Furthermore, the addition of DNA polymerase α and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase ε had been incubated with 3′-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3′-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3′→ 5′ exonuclease of human DNA polymerase ε can remove 3′-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."